caa a22 4500 397557256 CHVBK 20180308164806.0 cr unu---uuuuu 161202e199607 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021397 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021397 Conserved Histidine Residues of RCC1 Are Essential for Nucleotide Exchange on Ran [Elektronische Daten] [Yoshiaki Azuma, Hiroaki Seino, Takashi Seki, Satoru Uzawa, Christian Klebe, Tomoyuki Ohba, Alfred Wittinghofer, Naoyuki Hayashi, Takeharu Nishimoto] Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rccl− mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased Km, while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of Km similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence alanine scanning nationallicence mantGDP nationallicence Ran nationallicence RanGEF nationallicence RCC1 nationallicence Azuma Yoshiaki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut Seino Hiroaki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut Seki Takashi Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut Uzawa Satoru Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut Klebe Christian Abteilung Strukturelle Biologie, Max-Planck-Institut for Molekulare Physiologie Rheinlanddamm 201, 44139 Dortmund, Germany aut Ohba Tomoyuki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut Wittinghofer Alfred Abteilung Strukturelle Biologie, Max-Planck-Institut for Molekulare Physiologie Rheinlanddamm 201, 44139 Dortmund, Germany aut Hayashi Naoyuki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut Nishimoto Takeharu Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut The Journal of Biochemistry Oxford University Press 120/1(1996-07), 82-91 0021-924X 120:1<82 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021397 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021397 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Azuma Yoshiaki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Seino Hiroaki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Seki Takashi Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Uzawa Satoru Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Klebe Christian Abteilung Strukturelle Biologie, Max-Planck-Institut for Molekulare Physiologie Rheinlanddamm 201, 44139 Dortmund, Germany aut NATIONALLICENCE 700 1- Ohba Tomoyuki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Wittinghofer Alfred Abteilung Strukturelle Biologie, Max-Planck-Institut for Molekulare Physiologie Rheinlanddamm 201, 44139 Dortmund, Germany aut NATIONALLICENCE 700 1- Hayashi Naoyuki Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Nishimoto Takeharu Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/1(1996-07), 82-91 0021-924X 120:1<82 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford